Oestrogen and Antioestrogen Induced Gene Expression

The aim of this project was to study the effect of oestrogen and the antioestrogen tamoxifen on the expression of specific genes in the immature rat uterus and MCF-7 human breast cancer cells in order to try and further understand the mechanism of action of these two compounds. In order to do this a cDNA library was constructed using mRNA from 4 hour oestrogen-stimulated rat uteri. This was then screened with cDNA to mRNA from oestrogen stimulated and unstimulated rat uteri in order to isolate clones of oestrogen-regulated mRNAs. Twelve such clones were isolated and the expression of three of these, F4, B11 and E10, together with clones to a number of oncogenes, v-myc, v-Ha-ras, v-Ki-ras and c-sis, the oestrogen-regulated pS2 clone from MCF-7 cells, and an actin clone, p749, were studied in the immature rat uterus and MCF-7 cells in response to oestrogen and tamoxifen. In the immature rat uterus oestrogen caused a biphasic stimulation of expression of total mRNA with peaks at 4 and 16-20 hours after administration and, although the extent of induction was variable, it had a similar effect on all the clones studied, except actin which showed a continual increase from 0-20 hours after administration. Taking into account the slower uptake of tamoxifen, when compared to oestrogen, by uterine cells, and its metabolism to a more active derivative, this compound was found to be agonistic with respect to the induction of all the genes studied except the oestrogen-regulated clone from the rat uterine cDNA library, F4, which showed only one early peak of induction in response to tamoxifen. In MCF-7 human breast cancer cells oestrogen caused a 2-2.5 fold increase in mRNA. levels over control cells between 3 and 36 hours after administration, whereas tamoxifen treatment resulted in no increase in mRNA levels over the first 8 hours, and a decrease to half control level by 36 hours. Oestrogen also stimulated the expression of all the clones studied in this system, except the uterine library clone E10 which did not cross-react with MCF-7 cell RNA, though not to the same extent as in the immature rat uterus. However, the effect of tamoxifen on the amount of total mRNA available from MCF-7 cells meant that only two of the clones, pS2 and p-myc-2, could be studied in full. Of these the level of myc specific RNA was not increased at all but decreased steadily over the 36 hours studied, and although the level of pS2 specific RNA was increased within 1 hour of tamoxifen administration, this increase was only a fraction of that caused by oestrogen at 24 hours, and had fallen to control levels by 36 hours

Diet-induced obesity impairs mammary development and lactogenesis in murine mammary gland

We have developed a mouse model of diet-induced obesity that shows numerous abnormalities relating to mammary gland function. Animals ate 40% more calories when offered a high-fat diet and gained weight at three times the rate of controls. They exhibited reduced conception rates, increased peripartum pup mortality, and impaired lactogenesis. The impairment of lactogenesis involved lipid accumulation in the secretory epithelial cells indicative of an absence of copius milk secretion. Expression of mRNAs for -casein, whey acid protein, and -lactalbumin were all decreased immediately postpartum but recovered as lactation was established over 2–3 days. Expression of acetyl-CoA carboxylase (ACC)- mRNA was also decreased at parturition as was the total enzyme activity, although there was a compensatory increase in the proportion in the active state. By day 10 of lactation, the proportion of ACC in the active state was also decreased in obese animals, indicative of suppression of de novo fatty acid synthesis resulting from the supply of preformed fatty acids in the diet. Although obese animals consumed more calories in the nonpregnant and early pregnant states, they showed a marked depression in fat intake around day 9 of pregnancy before food intake recovered in later pregnancy. Food intake increased dramatically in both lean and obese animals during lactation although total calories consumed were identical in both groups. Thus, despite access to high-energy diets, the obese animals mobilized even more adipose tissue during lactation than their lean counterparts. Obese animals also exhibited marked abnormalities in alveolar development of the mammary gland, which may partially explain the delay in differentiation evident during lactogenesis

Induction of transcripts derived from promoter III of the acetyl-CoA carboxylase-alpha gene in mammary gland is associated with recruitment of SREBP-1 to a region of the proximal promoter defined by a DNase I hypersensitive site.

ACC-alpha (acetyl-CoA carboxylase-alpha), a key regulator of fatty-acid metabolism, is encoded by mRNAs transcribed from three promoters, PI, PII and PIII, in the ovine genome. Enhanced expression of transcripts encoded by PIII in mammary gland during lactation is associated with alterations in chromatin structure that result in the detection of two DNase I hypersensitive sites, upstream of the start site. The most proximal site, located between -190 and -10, is characterized by the presence of an inverted-CCAAT box, C2 at -167, and E-boxes, E1 and E2, at -151 and -46. Deletion of these motifs, which bind nuclear factor-Y and upstream stimulatory factors respectively in gel-shift assays, attenuates the activity of luciferase reporter constructs in transfected cells. Chromatin immunoprecipitation demonstrated that these transcription factors were associated with PIII in vivo in both lactating and non-lactating mammary tissues. The basic helix-loop-helix-leucine zipper transcription factor, SREBP-1 (sterol-regulated-element-binding protein-1), transactivated PIII reporter constructs in transfected HC11 mammary cells, and this was dependent on the presence of E1, but not on C2 or E2. SREBP-1 was only associated with PIII in chromatin from lactating animals, which was coincident with a 4-fold increase in the precursor (125 kDa) form of SREBP-1 in microsomes and the appearance of the mature form (68 kDa) in the nucleus. SREBP-1 motifs are also present in the proximal region of PII, which is also induced in lactation. This indicates that SREBP-1 is a major developmental regulator of the programme of lipid synthesis de novo in the lactating mammary gland